Enzyme expression methods and compositions

ABSTRACT

Methods for expressing active enzymes are described that involve co-expressing a first enzyme with a second enzyme that has an enzymatic activity that reverses a modification on the first enzyme and/or for identification of soluble and/or active catalytic domains by systematic variation of fragment lengths around catalytic domain boundaries.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. Nonprovisionalapplication Ser. No. 11/015,730, filed Dec. 16, 2004, now pending, whichclaims the benefit of U.S. Provisional Application 60/530,944, filedDec. 19, 2003, which is incorporated herein by reference in itsentirety, including drawings.

BACKGROUND OF THE INVENTION

The present invention relates to the field of expression of enzymes andfragments of enzymes.

For many applications, it is advantageous to express recombinantenzymes, which may be full-length enzymes or fragments of suchfull-length enzymes, e.g., catalytic domains. However, in some cases,expression of such an enzyme results in an unacceptable or variablelevel of modification of the enzyme, such as by self-modification or bymodification by host enzymes.

In addition, in some cases attempts to express a recombinant enzymeresults in insoluble and/or inactive enzyme. Such difficulties canarise, for example, when expressing eukaryotic coding sequences in aprokaryotic expression system such as E. coli.

SUMMARY OF THE INVENTION

The present invention concerns the provisions of polypeptides that havereduced levels of, or are free from, particular enzymatic modifications,especially attachment of modification moieties, and thus provides amethod for expressing an enzyme that has a reduced level of amodification moiety, such as phosphate groups. Such a method is usefulin a variety of different contexts. For example, phosphate modificationsare often involved in activation of enzymes that have a signaling orsignal amplification function, such as many different kinases. In othercases, a modification may occur at very high levels in the expressionsystem, which can interfere with normal activity and/or normalstructure. In still other cases, the presence of high modificationlevels may make it difficult to obtain crystals, or crystals may occurin an unnatural form due to the high density of the modifications, orcrystals may contain additional molecules or ions due to the presence ofadded modification moieties. In such cases, as well as others, it isbeneficial to reduce or even eliminate modification moieties of aparticular type or types.

The present invention accomplishes the reduction in modification byco-expressing the enzyme of interest with a second enzyme that reversesor counteracts the modification, e.g., removes the modification moiety.The expression level of the second enzyme can be established at adesired level to provide a desired level of reduction of modification ofthe first enzyme. A particular example of such enzyme pairing is apairing of kinase and phosphatase that have counteracting activities,e.g., protein tyrosine kinase and protein tyrosine phosphatase. In manycases, one or both of the enzymes is a fragment, e.g., a catalyticdomain, of a full-length protein.

Thus, in a first aspect, the invention concerns a method for expressinga recombinant kinase domain with reduced phosphorylation byco-expressing the recombinant kinase domain with a phosphatase domainthat removes phosphate groups from residues of the kinase domain.Typically the expression is in a cellular expression system.

In certain embodiments, the kinase domain and the phosphatase domain areexpressed separately from a single vector; the kinase domain and thephosphatase domain are expressed from a coding sequence as a fusionprotein (which can be joined with a cleavable linker to give separateproteins); the kinase domain and the phosphatase domain are expressedfrom a bi-cistronic mRNA; the kinase domain and the phosphatase domainare encoded by separate vectors; the kinase domain is from a proteintyrosine kinase; the phosphatase domain is from a protein tyrosinephosphatase; the kinase autophosphorylates; the kinase includes thehuman c-Met kinase domain; the kinase includes the human c-Abl kinasedomain; the co-expression results in reduced phosphorylation on thekinase as compared to expression of the kinase domain in the sameexpression system in the absence of a counteracting phosphatase; theco-expression results in improved crystallization of the kinase domainas compared to expression of the kinase domain in the same expressionsystem in the absence of a counteracting phosphatase; the co-expressionresults in a kinase preparation that has more uniform kinase activity ascompared to in the absence of such co-expression; the co-expressionresults in a kinase preparation that has increased specific activity ascompared to in the absence of such co-expression; the co-expressionresults in a kinase preparation that has improved activity as comparedto in the absence of such co-expression; the co-expression results in akinase having an increased drug inhibitor sensitivity (e.g., due to itshaving a state of modification giving it enzymatic and pharmacologicalproperties that mimics the state(s) that are the best targets for druginhibitors that can most effectively inhibit the enzyme to effect thedesired physiological response in the cell or tissue or organism).

As used in connection with production of defined polypeptides, the term“expressing” refers to the process of enzymatically synthesizing apolypeptide from a nucleic acid molecule that encodes the polypeptide.In most cases, the polypeptide is expressed in a cellular system.

Similarly, “co-expressing” refers to the expression of two differentpolypeptides concurrently in the same expression system, typically inthe same cellular expression system.

As used herein in connection with enzymes, the term “catalytic domain”refers to the portion of an enzyme where catalytic action occurs,delimited by amino acid sequence and three-dimensional structure. Thus,for a kinase, the term “kinase domain” refers to the catalytic domainwhere kinase activity is catalyzed. Persons skilled in the art arefamiliar with these terms, and catalytic domains have been identifiedfor many enzymes.

In connection with nucleic acid coding sequences and encodedpolypeptides, the term “recombinant” means that a nucleic acid sequencehas been removed from its natural sequence environment and inserted in adifferent environment and/or a nucleic acid sequence has been associatedwith different regulatory sequences such that expression from thenucleic acid sequence is significantly altered.

In connection with the activities of a protein modification enzymes andan enzyme that counteracts or reverses the activity of that enzyme, theterms “reverse” and “counteract” indicate that the enzyme thatcounteracts or reverses the activity of the modification enzyme reducesor eliminates the modification catalyzed by the modification enzyme in acommon substrate.

In the context of the present invention, the term “reduced modification”means that a particular enzymatic modification is present in substratemolecules, e.g., substrate proteins, at a lower level than in the sametype of substrate molecules under comparison conditions. For example,the level of modification present on substrate protein molecules can bereduced when the substrate proteins are expressed in the presence of anenzyme that reverses the modification. Thus, the term “reducedphosphorylation” refers to a lower level of phosphate groups (or eventhe absence of such groups) as compared to the level of phosphate groupspresent under comparison conditions.

In connection with enzymatic modification of proteins, the term“modification moiety” refers to a portion of a substrate protein that ischanged, typically by the addition or substitution of a chemical group,such as a phosphate group or methyl group.

In the context of protein expression, the term “cellular expressionsystem” refers to a system for expressing a protein in a cell duringculture. The protein product is typically recovered by lysing the cellsand purifying the desired product from the lysate.

As used herein, the term “bi-cistronic mRNA” refers to an mRNA that canbe translated to produce two independent polypeptide products (unlessspecifically indicated additional independent polypeptide products canalso be encoded by the mRNA such that the mRNA is “polycistronic”).

The term “vector” is used conventionally for molecular biology to referto a genetic construct that is adapted for insertion of recombinantnucleic acid sequences and transfection of cells to carry therecombinant nucleic acid sequence into the cells. Thus, “expressionvector” refers to a vector that is configured such that one or morepolypeptides are expressed from the vector intracellularly.

The terms “protein kinase” and “protein phosphatase” are usedconventionally to refer respectively to enzymes that catalyzephosphorylation and dephosphorylation of proteins. Thus, the terms“protein tyrosine kinase” refers to a protein kinase that catalyzesphosphorylation of protein substrates on tyrosine residues, while“protein tyrosine phosphatase” refers to a protein phosphatase thatremoves phosphate groups from tyrosine residues of substrate proteins.

In the context of the present invention, the term “self modify”indicates that a particular enzyme (which can be a catalytic domain)modifies molecules of that particular enzyme under suitable reactionconditions. Typically a molecule of the particular enzyme will modifyother molecules of that same particular enzyme; it can also typicallymodify other substrates. Thus, the term “autophosphorylate” refers to aself-modification in which a kinase enzyme phosphorylates molecules ofthat same kinase.

The term “endogenous enzyme” is used to refer to an enzyme that isnaturally produced in a cell. Thus, for example, in the context of thepresent invention, a kinase produced in a cell from a recombinantsequence may be phosphorylated by an endogenous protein kinase.

In the context of the present invention, the term “improvingcrystallization” indicates that the crystallizability and/or crystalquality of a polypeptide is improved under particular conditions ascompared to a reference condition. For example, a polypeptide that has auniform level of a chemical modification may crystallize more readilyand/or form better quality crystals (e.g., give higher resolutiondiffraction pattern) than a polypeptide that is heterogeneous withrespect to the level of that modification.

The phrase “more uniform activity” indicates that the enzymatic activityof repeat preparations is more consistent. The phrase “increasedspecific activity” refers to an increase in the enzymatic activity permilligram of the kinase protein in a preparation. The phrase “improvedactivity” refers to more uniform activity and/or increased specificactivity. In connection with activity of an enzyme with a changed levelor pattern of modification, the phrase ‘increased drug inhibitorsensitivity” refers to inhibition of the activity at concentrations of adrug inhibitor that would show less inhibition of a protein that ismodified differently.

In the context of modifications of a polypeptide in a proteinpreparation, the term “homogeneity” refers to the degree to which onlyone species of the polypeptide is present in the preparation. Thus, acompletely homogenous protein preparation will contain only one speciesof the particular polypeptide, e.g., a particular modification will bepresent to the same degree on all of that polypeptide in thepreparation.

As used herein, the term “substituent group” refers to a chemical groupattached on a molecule.

The term “fusion protein” is used herein in its conventional manner torefer to a protein that includes functional portions of at least twodifferent proteins in a single amino acid sequence. In many cases, thefused proteins (e.g., two fused proteins) are joined by a linkersequence that is not necessary and/or not involved in the activities ofthe fused proteins. Such a linker can be “cleavable”, meaning that thelinker can be broken under conditions such that functional separateprotein are produced from the fusion protein. In some cases, the linkercan be completely or at least largely removed, e.g., by cleaving thelinker at or near each end.

Reference to “Met” or “c-Met” herein refers to a transmembrane receptorbinding hepatic growth factor (i.e., hepatocyte growth factor receptor).This receptor is referred to as c-Met, because it is the normal cellularprotein that can malfunction to contribute to metastatic cancer.Specification of particular amino acid residues in c-Met utilizesequence NP_(—)000236, encoded by nucleotide sequence NM_(—)000245, atNCBI as the reference sequence.

Reference to “c-Abl” or “v-Abl” or “Abl” herein refers to Homo sapiensv-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1).Specification of particular amino acid resudyes in Abl utilize sequenceNP_(—)005148, encoded by NM_(—)005157 at NCBI.

In a related aspect, the invention provides a method for expressing in acell an enzyme having reduced enzymatic modification by co-expressing afirst recombinant enzyme with a second recombinant enzyme that removes amodification moiety from the first recombinant enzyme.

In particular embodiments, the first recombinant enzyme isself-modified; the first recombinant enzyme is expressed in a cell-basedexpression system and the first recombinant enzyme is modified by anendogenous enzyme produced by the cell; the first recombinant enzyme isselected from the group consisting of a kinase, a methylase, and anacetylase; the second recombinant enzyme is selected from the groupconsisting of a protein phosphatase, a protein demethylase, and aprotein deacetylase (selected to reverse a modification on the firstrecombinant enzyme).

In another related aspect the invention concerns a method for expressingenzymatically active c-Met kinase domain by co-expressing a c-Met kinasedomain with a phosphatase that removes phosphate groups from residues inthe c-Met kinase domain, whereby phosphorylation of the c-Met tyrosinekinase domain is reduced as compared to expression of the c-Met kinasedomain in the absence of expression of the phosphatase.

In particular embodiments, the c-Met kinase domain begins at one ofresidues 1049-1063 and ends at one of residues 1363-1408 of c-MET (theserange specifications include both the ranges and each individual residuewithin the ranges including endpoints) where particular embodimentsconcern each combination of starting residue and ending residue in thespecified ranges; the c-MET kinase domain consists essentially ofresidues 1056-1364; the c-Met kinase domain begins at one of residues1049-1063 and ends at a residue in a range of 1365-1370, 1371-1375,1376-1380, 1381-1385, 1386-1390, 1391-1395, 1396-1400, or 1401-1407(these range specifications include both the ranges and each individualresidue within the ranges including endpoints) where particularembodiments concern each combination of specific starting residue in thespecified range and specific ending residue in one of the specifiedranges.

Thus, in a related aspect, the invention provides polypeptide comprises,consists essentially of, or consists of a soluble c-Met kinase domain(e.g., human), for example, a human c-Met kinase domain. In particularembodiments, the c-Met kinase domain includes, consists essentially or,or consists of a polypeptide that begins at one of residues 1049-1063and ends at one of residues 1363-1408 or at a residue in the range of1365-1370, 1371-1375, 1376-1380, 1381-1385, 1386-1390, 1391-1395,1396-1400, or 1401-1407 of human c-Met, where additional particularembodiments are as specified in the preceding method.

In particular embodiments, the c-Met kinase domain consists essentiallyof residues 1056-1364 of c-Met; the polypeptide is free of phosphategroup modifications.

In another aspect, the invention provides a method for enhancingactivity of a recombinant enzyme expressed in an expression system wherethe enzyme is inactive when modified with a substituent group, byco-expressing the enzyme with a second recombinant enzyme, where thesecond recombinant enzyme removes those substituent group.

In another aspect, the invention provides a crystal of a purifiedpolypeptide consisting essentially of a protein modification enzymecatalytic domain, where the purified polypeptide self-modifies byaddition of a modification moiety, and the purified polypeptide is freeof that modification moiety.

In a related aspect, the invention provides a method for improvingcrystallization of a polypeptide subject to enzymatic modification byattachment of substituent groups when expressed in an expression system,by providing a purified polypeptide that has been co-expressed in theexpression system with an enzyme that reduces the level of or eliminatesmodification with the substituent groups, whereby the homogeneity of thepurified polypeptide is enhanced, and subjecting the purifiedpolypeptide to crystallization conditions.

In another aspect, the invention provides a nucleic acid sequenceencoding a first recombinant protein modification enzyme and a secondrecombinant protein modification enzyme that reverses the modificationcatalyzed by the first enzyme; and regulatory sequences adapted forexpression of the first and second enzymes in an expression system. Therespective coding sequences encoding the first and second enzymes may bein separate open reading frames, or in a single open reading frameencoding a fusion protein.

In a further aspect, the invention concerns a method for screening forenzyme inhibitors, by contacting a target enzyme free of a modificationwith a plurality of test compounds, where the target enzyme is activatedby the presence of at least one such modification, and determiningwhether any of the test compounds bind to or inhibit the enzymaticactivity of the target enzyme, where such binding or inhibition isindicative that the test compound is an inhibitor of the enzyme.

In particular embodiments, the target enzyme is a protein modificationenzyme that is obtained by coexpressing the target enzyme with a secondenzyme that reverses the protein self-modification catalyzed by thetarget enzyme; the target enzyme is a protein kinase, a methylase, or anacetylase; the target enzyme is obtained by coexpressing the targetenzyme with a second enzyme that removes activating modificationmoieties on the target enzyme.

As used herein in connection with enzyme activity, the term “active”indicates that the polypeptide gives a signal that is at least 2-foldthe background signal for the protein in a standard assay accepted bythose of skill in the art for the relevant enzyme. For example, inprotein kinase enzyme assays the minimal readout used to demonstrateactivity is two-fold above the background readout created when the assayprotocol is performed in the absence of the substrate ATP. In the caseof a protein tyrosine kinase, antibodies are available (e.g., PY20,Perkin Elmer) that bind tightly to phospho-tyrosine residues that arethe product of the enzyme reaction. Sensitive light signals of thisantibody binding can be created using the AlphaScreen instrument (PerkinElmer), from which the amount of activity of the kinase can be inferred.In the absence of the above assay, other standard protein kinase assayformats, such as the common ELISA format, also can be used to detectactivity. In the ELISA format the substrate protein can be attached tothe bottom of assay plates, and after the enzyme reaction is performedantibodies such as PY20 are bound to the phospho-tyrosine residuesproduced. Secondary antibodies that are attached to horseradishperoxidase and that bind the PY20 antibody can be used to produce acolor reaction when reacted with specific substrates such as TMB(3,3′,5,5′-tetramethylbenzidine). The amount of light absorbance causedby the bound horseradish peroxidase will relate to the amount ofphospho-tyrosine residues present, from which the activity of the kinasecan be inferred. Suitable assays are also known in the art for otherenzymes, such as other protein kinases (e.g., serine/threonine andhistidine kinases) and other enzymes. As the converse to the term“active”, the term “inactive” indicates that the protein produces lessthan 2-fold signal over background in the relevant standard enzymeassay.

The term “activate”, in connection with an enzyme, indicates that theenzyme has been changed in a manner resulting in significantly increasedactivity on a suitable substrate. In some cases, the activity of thenon-activated form of the enzyme can be undetectable. In many cases,phosphorylation of a specific residue results in activation, e.g.,activation of a kinase.

As used herein in connection with modulators of activity of a, the term“screening” refers to determining whether any of a plurality of testcompounds have a desired biological effect, such as inhibition of anactivity of a particular protein. The plurality of compounds, can forexample, be at least 10, 100, 1000, 10,000, or more.

In yet another aspect, the invention concerns an expression vector thatincludes a first recombinant nucleic acid sequence encoding a firstenzyme subject to enzymatic modification and a second recombinantnucleic acid sequence encoding a second enzyme that reverses thatmodification, where the first and second recombinant nucleic acidsequences are operatively linked with regulatory sequences such that thefirst and second recombinant nucleic acid sequences are expressed in ahost cell.

A related aspect concerns a cell that includes a first recombinantnucleic acid sequence encoding a first enzyme subject to enzymaticmodification, and a second recombinant nucleic acid sequence encoding asecond enzyme that reverses that modification, where the first andsecond recombinant nucleic acid sequences are operatively linked withregulatory sequences such that the first and second recombinant nucleicacid sequences are expressed in the cell.

In certain embodiments of the present invention that involveco-expression of enzymes or constructs for carrying out such expression,expression is carried out in a cell-based expression system; thesubstituent group or modification moiety is a phosphate group, methylgroup, or acetyl group; one or both of the first and second enzymes areprovided as catalytic domains, such as kinase domain, phosphatasedomain, methylase domain, de-methylase domain, acetylase domain,de-acetylase domain; a kinase domain is a c-Met kinase domain; an enzymepair is a protein kinase and a protein phosphatase (e.g., a tyrosineprotein kinase and a protein tyrosine phosphatase), a methylase and ade-methylase, or an acetylase and a de-acetylase; sequences encoding thefirst and second enzymes are configured such that the first and secondenzymes will be or are encoded by a single mRNA, i.e., a bi-cistronicmRNA; sequences encoding the first and second enzymes are configuredsuch that the first and second enzymes will be or are encoded byseparate mRNAs; the first and second enzymes are expressed from avector; the first enzyme is a protein modification enzyme; the firstenzyme self-modifies when expressed as a recombinant protein in abacterial cell, such as in E. coli; the first enzyme consistsessentially of a kinase domain and the second enzyme consistsessentially of a phosphatase domain that reduces the level of phosphategroup modification on the kinase domain; the first enzyme and the secondenzyme are expressed at levels such that the ratio of first enzyme tosecond enzyme is in the range of 1:10 to 10:1, 1:5 to 5:1, 1:3 to 3:1,1:2 to 2:1, or 1.5:1 to 1:1.5; sequences encoding the first and secondenzymes are linked by additional coding sequences such that a singlefusion protein having both enzymes is produced; first and second enzymesin a fusion protein are linked by additional coding sequences thatfunction as a cleavable linker; the first and/or second enzymes areproduced from genes stably integrated with the genes of the expressioncell.

In another aspect, the invention concerns a method for identifying asoluble enzyme fragment, where the method involves expressing a set offragments of the enzyme, thereby determining solubility and/or enzymaticactivity of a plurality of fragments. One or more of the fragments areselected that provide soluble and/or active protein. If none of thefragments are soluble and/or active, a new set with different terminiare constructed and tested until one or more fragments are identifiedthat provide soluble and/or active enzyme. Generally, the process iscarried out systematically such that available information about theenzyme is utilized in selecting termini for the fragments, such asexclusion of sequences that would result in surface hydrophobic residuepatches, inclusion of sequences representing all or most of thecatalytic domain.

In particular embodiments, at least 5, 7, 10, or more fragments of anenzyme that is insoluble when expressed in catalytic domain-length formare expressed and tested; each of the fragments has a C-terminal aminoacid residue that is in the range of 100 residues outside to 20 residuesinside the C-terminal catalytic domain boundary (or 100 residues outsideto 10 residues inside, 70 residues outside to 10 residues inside, or 70residues outside to 5 residues inside), and/or an N-terminal amino acidresidue that is in the range of 100 residues outside to 20 residuesinside the N-terminal catalytic domain boundary or 100 residues outsideto 10 residues inside, 70 residues outside to 10 residues inside, or 70residues outside to 5 residues inside); and analyzing relative levels ofsoluble and/or active polypeptide for each fragment in said set, therebyidentifying soluble and/or active enzyme fragments, if any, in the set.

As used herein in connection with an enzyme, the term “set of fragments”refers to a plurality of portions of the amino acid sequence of theenzyme that include a sequence portion in common but have differenttermini at the C-terminus, N-terminus, or both.

In certain embodiments, the method is performed by analyzing a firstplurality of fragments each having the same C-terminal amino acidresidue and differing in their N-terminal amino acid residues for levelsof soluble enzyme; selecting a fragment that provides soluble enzyme,where the fragment has a selected N-terminal amino acid residue;analyzing a second plurality of fragments that each has the selectedN-terminal amino acid residue; and selecting a fragment that provides ahigh level of soluble enzyme relative to other fragments in said secondplurality, The selected C-terminal amino acid residue in the firstplurality of fragments can be selected to be at any of a variety oflocations, for example, within 10, 20, 30, 40, or 50 residues of theC-terminal residue of the full-length enzyme polypeptide that includesthat fragment and/or at least 10, 20, 30, 40, 50, 60, 80, or 100residues outside the C-terminal catalytic domain boundary.

In particular embodiments, the method is carried out by analyzing afirst plurality of fragments each comprising the same N-terminal aminoacid residue and differing in their C-terminal amino acid residues forlevels of soluble enzyme; selecting a fragment that provides solubleenzyme, wherein the fragment has a selected C-terminal amino acidresidue; analyzing a second plurality of fragments that each has thatselected C-terminal amino acid residue and different N-terminal aminoacid residues; and selecting a fragment that provides a high level ofsoluble enzyme relative to other fragments in the second plurality. Asindicated above for the C-terminus the N-terminus can be selected to beat a variety of different locations, for example, the N-terminal aminoacid residue in the first plurality of fragments is within 10, 20, 30,40, or 50 residues of the N-terminal residue of the full-length enzymepolypeptide that includes the fragment and/or the N-terminal amino acidresidue in the first plurality of fragments is at least 50 residuesoutside the N-terminal catalytic domain boundary

As used in connection with expression of complete or partial enzymes,the term “enzymatically active” indicates that the enzyme (e.g.,catalytic domain) catalyzes the corresponding reaction on a substrateappropriate for that enzyme with an activity level that is detectableusing an assay suitable for that enzyme. Conversely, the term “inactive”indicates that the enzyme catalyzes the corresponding reaction on asubstrate appropriate for that enzyme at a level that is not detectableusing the same assay.

In connection with expression of a recombinant polypeptide, the term“soluble” indicates that the polypeptide will remain in solution underparticular conditions, i.e., does not precipitate out of solution orform large suspended clumps. In the context of the present invention,the term typically refers to solubility in an aqueous solution, e.g.,one suitable for carrying out an enzymatic reaction with thepolypeptide.

Additional embodiments will be apparent from the following DetailedDescription and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the tyrosine kinase domain within human c-MET, andthe various fragments constructed in an exemplary demonstration ofidentification of soluble catalytic domain from an insoluble parentmolecule.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Genetic engineering methods can enable a person to produce amounts ofgene-encoded proteins much higher than exist in normal organisms. Whensuccessful, such material can allow research and development related tothese proteins that would be difficult or impossible using only materialisolated from the normal organism. However, often the material producedthrough genetic engineering has inadequate yield, or fails to haveproperties expected of material from the normal organism, and suchdifferences can limit their utility.

In the case of protein-modification enzymes, failure can result from anumber of different causes. For example, the enzyme may alter theproteins of the expression host sufficiently to cause lower levels ofenzyme production, such as through toxic effects on the host. Whenhigher production levels are achieved, the enzyme may alter thestructure of itself in an unnatural manner due to the high productionlevels, leading to alterations in normal properties or even completeloss of activity.

In addition to the difficulties involved in expression of recombinantenzymes in substantial quantities, some proteins, e.g., catalyticdomains of enzymes, are difficult to obtain in active, soluble form.Examples include kinase domains of a number of membrane bound proteinkinases, e.g., receptor tyrosine kinases.

The present invention provides methods for addressing both of thedifficulties discussed above.

Co-Expression of Enzymes Having Counteracting Activities

Methods described here involve producing the protein modification enzymeof interest coordinately with a second protein or agent having activitythat can reverse or limit the unwanted actions. In a particular example,a protein tyrosine kinase (transferring phosphate from adenosinetriphosphate to tyrosine groups on proteins) is simultaneously producedin bacteria with a protein tyrosine phosphatase (cleaves phosphate fromphosphotyrosine groups on proteins). When produced singly, the proteintyrosine phosphatase becomes hyperphosphorylated, whereas when producedsimultaneously with a protein tyrosine phosphatase it is essentiallyunphosphorylated.

The ability to produce the dephosphorylated kinase is advantageous forpharmaceutical research because this form allows biochemical assays tobe performed that are impossible with the hyperphosphorylated form. Forexample, currently the sole kinase inhibitor approved for use in humantherapy is4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamidemethanesulfonate (Gleevec), which is known to target thedephosphorylated kinase specifically. In the biology of kinases,activity is often stimulated by residue-specific phosphorylation of thekinase by a second kinase or by residue-specific autophosphorylation,after which the kinase is in an activated state.

Thus, in at least some cases, the most effective kinase-inhibitorypharmaceuticals will act on the unphosphorylated kinase before it isactivated, because the unactivated kinase may be more easily controlledif it is inhibited before it can be activated. In such cases, providinga source of unphosphorylated kinase is valuable for kinasepharmaceutical research and development. In situations where researchand/or development with the phosphorylated, activated kinase is desired,having the unphosphorylated kinase is also valuable as a startingmaterial for creating specifically phosphorylated protein undercontrolled conditions, without hyperphosphorylation at incorrectresidues.

In addition to allowing use of non-activated enzyme in cases where amodification such as a phosphorylation activates the enzyme, reducing oreliminating a modification on a target enzyme can also be applied inother cases where the presence of a modification or modifications causesa difference in sensitivity of the target enzyme to modulator compounds,e.g., small molecules. For example, the presence of a modification(s)can alter the binding characteristics for binding of modulator in abinding site, e.g., at an active site. Such binding characteristics canoccur by various mechanisms, e.g., by steric hindrance or blocking,preventing the modulator compound from binding, and/or by inducingcomformational changes in the protein that alter the binding site in amanner that changes binding properties for modulators. Thus, in avariety of applications, it is beneficial to use protein for screeningthat is free from, or has a reduced level of modification, e.g. proteinproduced using co-expression as described herein. Such protein can beused directly, or can be subjected to controlled modification tointroduce a desired level of modification or modification at specificsites. In many cases, the unmodified form is more sensitive toinhibitors.

Producing unmodified enzymes (e.g., dephosphorylated kinase) is alsoadvantageous in the application of X-ray crystallography forpharmaceutical research. X-ray crystallography requires a source oftarget protein that can be purified to near-homogeneity so that thecrystals obtained will yield high-resolution X-ray diffraction data. Ifa protein modification enzyme acts on itself, it will then exist inmultiple forms (with different modification levels) that are difficultto purify to homogeneity. Producing unmodified enzyme, e.g., producingthe unphosphorylated form of a protein kinase, as a homogeneous startingform is a distinct advantage. As with biochemical assays, the unmodifiedmaterial can be used for structure analyses directly or after activatingmodification reactions have been performed under controlled conditions.

The method is not limited to kinases and phosphatases, but instead canbe applied to the design of expression systems for numerous types ofprotein modification enzymes including, for example, without limitation:

a) protein kinase with protein phosphatase

b) protein methylase with protein demethylase

c) protein acetylase with protein deacetylase

Although, in many cases, coexpression with the second enzyme is intendedprimarily to reverse or limit the activity of the first enzyme acting onitself, situations can also arise where the expression host or systemhas activities that modify the first enzyme. Such host modification canlikewise be reversed by coexpression with the second enzyme. Forexample, an expression host system (e.g., a bacterial expression system)can modify the first enzyme in an unnatural manner, and those unnaturalmodifications can be removed with a co-expressed enzyme that removes theparticular modification.

To achieve coordinate production of a protein modification enzyme with areversing agent the two genes can be engineered together on a single DNAfragment, such as on a prokaryotic plasmid, either as two separate genesor such that a polycistronic mRNA is produced, or such that a fusionprotein is produced. Alternatively the two genes can be engineered ontwo separate DNA fragments. The two genes may be differently localizedor regulated in the cell, and introduced into the cell or organism atdifferent times or by different means. The levels of proteinaccumulation and levels of activity of the protein modification enzymeand reversal agent encoded by the two genes may be varied separately toachieve different amounts of protein modification reversal. Thereversing agent might be produced by a separate process and introducedinto the cell or organism as a mRNA or protein rather than as a gene.Further, either or both of the protein modification enzyme and thereversing agent might be produced in an in vitro translation process.

After the production period, any of numerous schemes can be utilized formanipulating the protein modification enzyme to remove the reversingagent as needed, e.g., using a tag that allows the reversing agent to bepreferentially bound to a solid phase medium and thereby separated fromthe protein modification enzyme.

Production of Soluble Enzyme Domains

As indicated above, when a person works with a protein or proteindomain, usually an enzyme catalytic domain, in some cases difficultiesare experienced in producing soluble active protein. For example, duringeither production in a cell or during purification, much (or evenessentially all) of the recombinant protein agglomerates and/orprecipitates, resulting in little or no soluble active recombinantprotein.

It was found that often this problem can be solved by proper selectionof the termini of the recombinant protein. In the case of an enzymecatalytic domain, it is common to attempt to use a protein fragment withtermini at or close to the boundaries of the catalytic domain.

We discovered that the exact termini utilized can have a significant,even dramatic, effect on the level of production and/or activity ofsoluble recombinant protein. In some cases, varying a terminus by evenone amino acid residue can significantly alter the amount of solubleand/or active protein obtained.

Thus, the present method involves systematic testing of multiplefragments to identify suitable termini for a soluble and/or activeprotein. This process can be performed in various ways, but generallyinvolves testing a plurality of fragments while co-varying the terminiin small increments around the N- and C-termini of the domain ofinterest (or other portion of interest of the protein). One or morespecific fragments are selected that give levels of soluble and/oractive protein that are better than others in the set. One or more ofthese fragments then serves as the basis for selecting and testingadditional fragment that have termini close to the termini of theinitially selected fragment. Such selection and testing can be performedmultiple times as needed, e.g., using small variations. If needed toobtain acceptable production, one or both termini can be varied toproduce a set of fragments that differ by as little as 1 residue at aterminus (or both termini).

In practice, it is often advantageous to vary only one terminus at atime. In such an implementation, one terminus is fixed (e.g., at adomain boundary or at the end of the native protein) while the otherterminus is varied to produce a set of fragments that vary in length bysmall increments. This initial set is tested. If a fragment or fragmentprovides better production than others, such fragment or fragments canbe selected as the basis for a second round of construction andselection. While the same terminus can be varied on a finer scale, inmany cases, it is more efficient to vary the other (previouslyinvariant) terminus, test the resulting fragments, and select a fragmentthat gives better production. This process can be repeated, varying oneor both termini to identify a fragment that provides acceptableproduction.

The selection of initial termini can depend on where in the nativeprotein the domain (or other portion of interest) is located. Forexample, if the domain is located near one end of the native protein, itcan be advantageous to initially fix one terminus at or near the end ofthe native protein, and vary the other terminus. If the domain islocated further away from the native terminus, it can be advantageous toutilize an initial terminus outside the domain on one end, and vary theother terminus near the other domain boundary.

In many cases, a terminus will be varied within the range of 100 aaoutside to 20 aa inside a domain boundary (e.g., catalytic domainboundary). In other embodiments, one or both termini can be varied inthe range of 100 aa outside to 10 aa inside, 50 outside to 20 inside, 50outside to 10 inside, 100 outside to 5 inside, 50 outside to 5 inside,30 outside to 20 inside, 30 outside to 10 inside, 30 outside to 5inside, 100 outside to 0 inside, 50 outside to 0 inside, 30 outside to 0inside a domain boundary, as well as other ranges.

Nucleic acid sequences encoding protein or polypeptides for use in thepresent invention can be obtained from conventional sources, e.g., fromsequence depositories, synthesized from sequence information, subclonedfrom a clone library, or cloned from a source organism.

Genetic constructs useful in the present invention can be constructedusing conventional cloning methods, allowing cloning, construction ofrecombinant constructs, production and purification of recombinantprotein, introduction of constructs into other organisms, and othermolecular biological manipulations of specific protein coding sequencesare readily performed.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well disclosed in the scientific and patent literature,see, e.g., Sambrook, ed., Molecular Cloning: a Laboratory Manual (2nded.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CurrentProtocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc.,New York (11997); Laboratory Techniques in Biochemistry and MolecularBiology: Hybridization With Nucleic Acid Probes, Part T. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993), as well asnumerous other sources.

Nucleic acid sequences can be amplified as necessary for further useusing amplification methods, such as PCR, isothermal methods, rollingcircle methods, etc., are well known to the skilled artisan. See, e.g.,Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al.,Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam et al.,Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner et al.,Biotechniques 2001 April; 30(4):852-6, 858, 860 passim; Zhong et al.,Biotechniques 2001 April; 30(4):852-6, 858, 860 passim.

Nucleic acids, vectors, capsids, polypeptides, and the like can beanalyzed and quantified by any of a number of general means well knownto those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatography, various immunological methods, e.g. fluidor gel precipitin reactions, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, Southern analysis, Northern analysis,dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid ortarget or signal amplification methods, radiolabeling, scintillationcounting, and affinity chromatography.

Obtaining and manipulating nucleic acids used to practice the methods ofthe invention can be performed by cloning from genomic samples, and, ifdesired, screening and re-cloning inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in.e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

The nucleic acids of the invention can be operatively linked to apromoter. A promoter can be one motif or an array of nucleic acidcontrol sequences which direct transcription of a nucleic acid. Apromoter can include necessary nucleic acid sequences near the startsite of transcription, such as, in the case of a polymerase II typepromoter, a TATA element. A promoter also optionally includes distalenhancer or repressor elements which can be located as much as severalthousand base pairs from the start site of transcription. A“constitutive” promoter is a promoter which is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter which is under environmental or developmental regulation. A“tissue specific” promoter is active in certain tissue types of anorganism, but not in other tissue types from the same organism. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

Depending on the host vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, may be used in the expression vector. Forexample, when cloning in bacterial systems, inducible promoters such aspL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used; when cloning in insect cell systems, promoterssuch as the baculovirus polyhedrin promoter may be used; when cloning inplant cell systems, promoters derived from the genome of plant cells(e.g., heat shock promoters; the promoter for the small subunit ofRUBISCO; the promoter for the chlorophyll a/b binding protein) or fromplant viruses (e.g., the 35S RNA promoter of CaMV; the coat proteinpromoter of TMV) may be used; when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used;when generating cell lines that contain multiple copies of the desiredDNA, SV4O-, BPV- and EBV-based vectors may be used with an appropriateselectable marker.

Thus, the nucleic acids of the invention can be provided in expressionvectors and cloning vehicles, e.g., sequences encoding the polypeptidesof the invention. Expression vectors and cloning vehicles of theinvention can comprise viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors can include chromosomal, non-chromosomal and synthetic DNAsequences. Large numbers of suitable vectors are known to those of skillin the art, and are commercially available.

The nucleic acids of the invention can be cloned, if desired, into anyof a variety of vectors using routine molecular biological methods;methods for cloning in vitro amplified nucleic acids are disclosed,e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of amplifiedsequences, restriction enzyme sites can be “built into” a PCR primerpair. Vectors may be introduced into a genome or into the cytoplasm or anucleus of a cell and expressed by a variety of conventional techniques,well described in the scientific and patent literature. See, e.g.,Roberts (1987) Nature 328:731; Schneider (1995) Protein Expr. Purif6435:10; Sambrook, Tijssen or Ausubel. The vectors can be isolated fromnatural sources, obtained from such sources as ATCC or GenBanklibraries, or prepared by synthetic or recombinant methods. For example,the nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses which are stably or transiently expressedin cells (e.g., episomal expression systems). Selection markers can beincorporated into expression cassettes and vectors to confer aselectable phenotype on transformed cells and sequences. For example,selection markers can code for episomal maintenance and replication suchthat integration into the host genome is not required.

A variety of host-expression vector systems may be utilized to expressthe desired coding sequence(s). These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the coding sequence; yeast transformed with recombinant yeastexpression vectors containing the coding sequence; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing the coding sequence; oranimal cell systems. The expression elements of these systems vary intheir strength and specificities.

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a polypeptide ofthe invention, or a vector of the invention. The host cell may be any ofthe host cells familiar to those skilled in the art, includingprokaryotic cells, eukaryotic cells, such as bacterial cells, fungalcells, yeast cells, mammalian cells, insect cells, or plant cells.Exemplary bacterial cells include E. coli, Streptomyces, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art.

Vectors may be introduced into the host cells using any of a variety oftechniques, including transformation, transfection, transduction, viralinfection, gene guns, or Ti-mediated gene transfer. Particular methodsinclude calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation.

Engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the invention. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter may be induced byappropriate means (e.g., temperature shift or chemical induction) andthe cells may be cultured for an additional period to allow them toproduce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the polypeptide. If desired, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

In addition to bacterial culture systems, various mammalian cell culturesystems can also be employed to express recombinant protein. Examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts and other cell lines capable of expressing proteins from acompatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Exemplary embodiments of the invention are shown by the non-limitingexamples described herein.

EXAMPLES Example 1 Engineering a Bicistronic Vector Encoding Both AblKinase Domain and PTP1b Phosphatase Domain

The bicistronic vector engineering used, as a starting material, apreviously-engineered vector that encodes human Abl (Abelson tyrosinekinase) kinase domain extending from Abl residue Gly 227 through toresidue Val 515, termed pET-SPEC BI-PTP Abl G227-V515-X. The section ofthis plasmid that encodes Abl kinase and PTP1b domains on a bicistronicmRNA transcript is shown in Table 2. The remainder of the vector is aderivative of the pET-24 vector (Novagen) designed to utilize the T7 RNApolymerase for producing mRNA in strains of E. coli that are engineeredto produce that polymerase.

A DNA fragment encoding the human PTP1b phosphatase domain extendingfrom PTP1b residue Met 1 through to residue Gly 283 was engineered usingPCR. The template for the PCR reaction was a previously-engineered humanPTP1b cDNA derived from a purchased total human brain cDNA library(Invitrogen). Custom oligonucleotide primers (Invitrogen) were designedto create a ribosome binding site and a SalI site (GTCGAC) flanking the5′-side (PTP-SAL), and an EcoRI site (GAATTC) flanking the 3′-side(PTP-RI) of the encoded phosphatase domain. The PTP-SAL oligonucleotideprimes the coding strand of PTP1b and the PTP-RI PCR oligonucleotideprimes the non-coding strand of PTP1b. These primer sequences are shownbelow:

DNA oligonucleotide primer - Coding strand PTP-SAL 5′-CTGCGAA GTCGACGAAGGAGATATATCC ATGGAGATGGAAAAGGAGTTCG (SEQ ID NO: 1) DNAoligonucleotide primer - Noncoding strand PTP-RI 5′-CTGCGAAGAATTCTCACCCCATGATGAATTTGGCACCT (SEQ ID NO: 2)

After performing a standard PCR reaction, the product obtained was cutwith the restriction enzymes, SalI and EcoRI (New England Biolabs), andpurified by agarose gel electrophoresis. The pET-SPEC Abl G227-V515-Xvector contains unique SalI and EcoRI restriction sites immediatelydownstream of the Abl coding region. The pET-SPEC Abl G227-V515-X vectorwas cut with SalI and EcoRI, and purified by agarose gelelectrophoresis. The PTP1b DNA and the pET-SPEC Abl G227-V515-X vectorDNA were ligated together using T4 DNA ligase (Invitrogen) to create acircular plasmid suitable for the bicistronic expression of the Ablkinase domain and the PTP1b phosphatase domain in E. coli.

The plasmid vector was introduced into E. coli, amplified during the E.coli growth, and extracted for analysis. Relevant DNA sequences weredetermined (Davis Sequencing). The relevant DNA sequence for thisvector, pET-SPEC BI-PTP Abl G227-V515-X, with the encoded amino acidsequences of the Abl and PTP proteins, shown in Table 2. The DNAsequence that matches the mRNA transcript is shown above the amino acidsequences that are encoded by this mRNA. The bicistronic mRNA encodesfirst the Abl kinase followed by the PTP1b phosphatase domain. Ablkinase domain extends from residue G227 through V515, and is preceded byan N-terminal His tag (MGHHHHHH). (SEQ ID NO: 3) The PTP1b phosphatasedomain extends from residue M1 through G283. Both the Abl and PTP1bcoding regions are preceded by ribosome binding sites (AAGGAG) in themRNA.

Example 2 Expression in E. coli of his-Tagged Abl Protein from pET-SPECBI-PTP Abl G227-V515-X

To obtain protein expression of dephosphorylated Abl, the pET-SPECBI-PTP Abl G227-V515-X DNA was transfected into the E. coli strainBL21-CodonPlus(DE3)-RIL (Stratagene) using standard methods, with dualantibiotic selection of kanamycin and chloramphenicol. E. coli harboringthe plasmid were grown in liquid culture at 37° C. with shaking to anOD600=1, at which point the culture temperature was reduced to 15° C.,and the 0.5 mM IPTG inducing agent was added. The culture was thenshaken for 18 hrs at 15° C., at which point the E. coli was concentratedby centrifugation.

The E. coli pellet, 0.5 g, was suspended in 10 ml buffer (50 mM Tris pH7.5, 250 mM NaCl, 0.1% Triton-X-100, 0.02% monothioglycerol, 20 uMphenylmethylsulfonyl chloride). Extraction was initiated by addition oflysozyme (Sigma) to 200 ug per ml, incubation on ice for 15 min, andsonication for 1 min. Solution was centrifuged 30 min at 17000 RPM inSA600 rotor (Sorvall) at 4 C. The supernatant was recovered, andHistidine-tagged Abl protein was purified using metal affinitychromatography by mixing supernatant with 600 ul 50% slurry ofbuffer-washed Talon beads (Clontech) for 1 hr at 4° C., in the presenceof 10 mM imidazole. The beads are washed 3 times with 10 mls of buffer(50 mM Tris pH 7.5, 100 mM NaCl, 0.02% monothioglycerol, 20 uMphenylmethylsulfonyl chloride and 10% glycerol) with centrifugation at4000 RPM between washes. Abl protein was eluted from pelleted beadsusing 1 ml buffer (50 mM Tris pH 7.7, 100 mM NaCl, 10% glycerol, 200 mMimidazole). Eluted proteins were concentrated by centrifugation inCentriprep concentrators (Millipore).

The concentrated protein was buffer-exchanged by gel filtrationchromatography using P-10 columns (Pharmacia), equilibrated in buffer(50 mM Tris pH 7.7, 100 mM NaCl, and 10% glycerol). Eluted protein wasflash-frozen in liquid nitrogen and stored at −80° C. With thispurification protocol His-tagged Abl protein at >80% purity is achieved.The coexpressed PTP is not present, as expected because it lacked theHistidine tag and should therefore not bind the Talon beads.

Example 3 Determination that Abl Co-Expressed with Phosphatase is notPhosphorylated

Abl proteins expressed either with or without PTP coexpression wereevaluated for state of phosphorylation detectable with an antibody thatspecifically binds phospho-Tyr (PY20 mouse monoclonal IgG_(2b),SantaCruz Biotechnology). Samples of Abl made from each expressionvector were separated by size using SDS-PAGE, then transferred byelectro-blotting onto Imobilon-P membrane (Millipore) in transfer buffer(39 mM Glycine, 48 mM Tris HCl, 20% MeOH, 0.0375% SDS). The membrane wasstained with coomassie to visualize the Abl proteins. Before exposure toantiserum the membrane was exposed to phosphate-buffered salinecontaining 0.1% Tween detergent and 5% bovine serum albumin to reducebackground antibody binding. The paper was then exposed for 2 hr at roomtemperature to 1:1000 dilution of PY20 antibody prepared in the samebuffer. After washing, membrane was exposed for 2 hr at room temperatureto 1:5000 dilution goat anti-mouse IgG (H+ L) coupled to horse-radishperoxidase (Pierce). Membrane is washed three times as before.Visualization of binding was made using chemical luminescencemethodology with ECL detection reagent (Amersham Biosciences) accordingto manufacturer's protocol. Inspection of the finished blot indicatedthat the Abl coexpressed with PTP1B has little or no phosphorylationabove background, whereas Abl coexpressed alone is highlyphosphorylated.

Example 4 Determination of Activities of Unphosphorylated Abl andHyperphosphorylated Abl

The activity of the Abl was assayed using AlphaScreen Phosphotyrosine(PY20) Assay Kits (PerkinElmer). 20 ng of Abl protein was mixed with 5uM ATP (Sigma), 50 pmol BIO-E4Y3 peptide (New England Biolabs), 20 nlStreptavidin donor beads, and 20 nl Anti-phosphotyrosine (PY20) acceptorbeads in 20 ul reaction buffer (50 mM Hepes pH 7.1, 1 mM MgCl₂, 0.1%IGPAL, 0.005% BSA), and incubation performed at 37° C. for 2 hoursbefore reading in a Fusion Universal Microplate Analyzer (Perkin Elmer).For inhibitor studies, 1 ul of drug was added to the empty assay platefirst.

The Abl coexpressed with PTP was compared to Abl expressed alone forsensitivity to the inhibitor, Gleevec. Various concentrations weretested for inhibition of the kinase signal seen in the AlphaScreenassay. Gleevec could inhibit completely both types of Abl, but 47 nMGleevec was required to achieve 50% inhibition of the Abl coexpressedwith PTP, whereas 5 uM Gleevec achieved 50% inhibition of the Ablexpressed alone, indicating that 100-fold higher Gleevec was required toinhibit the phosphorylated form.

Example 5 Engineering a Family of Bicistronic Vectors for Coexpressionof Either Yersinia tyrosine Phosphatase, VHR Phosphatase, or LambdaPhosphatase

Phosphatases vary in their substrate specificities and activities. PTP1bis restricted to phosphotyrosine as substrate, whereas otherphosphatases act on other phosphorylated residues, e.g.,serine/threonine or histidine. Thus, other selections of phosphatasescan be used in particular applications. To expand the choices ofphosphatase for coexpression, vectors were engineered having YOPtyrosine phosphatase, VHR phosphatase, or lambda phosphatase. Sequencesfor other types of phosphatases (as well as other enzymes) can beobtained from publicly available sequence databases, such as GenBank,SwissProt, and the like. For example, the amino acid sequence andaccession number for lambda phosphatase is provided in Table 4.

For engineering of the YOP and lambda vectors, PCR cloning methodsanalogous to the engineering of the bicistronic vector for coexpressionwith PTP1B were used. For the VHR vector, a complete gene synthesis ofthe VHR coding region was performed using synthetic oligo primers. Eachvector has NdeI and SalI sites available for accepting DNA fragmentsencoding targets of interest as a Histidine-tagged protein on a singlemRNA also encoding the non-tagged phosphatase. We refer to these vectorsas pET-N6 BI-YOP, pET-N6 BI-VHR, and pET-N6 BI-LAM.

Example 6 Engineering a Soluble, Enzymatically Active c-Met KinaseDomain by Systematically Varying the Choice of Encoded N-Terminal andC-Terminal Kinase Domain Boundaries

The MET kinase domain is the intracellular part of a transmembranereceptor binding hepatic growth factor (i.e., hepatocyte growth factorreceptor). This receptor is referred to as c-Met, because it is thenormal cellular protein that can malfunction to contribute to metastaticcancer. Although of interest for 20 years as a target for anti-cancerdrug development, it has never successfully been expressed as a solubleprotein in E. coli. We identified boundaries of the MET kinase domainthat yield soluble active protein when engineered for expression inbacteria.

The first step of a two-step approach was to systematically test severaldifferent upstream boundaries in conjunction with the natural C-terminus(residue 1408), and to examine the E. coli-expressed products for thecomparative levels of soluble MET kinase protein and comparative levelsof insoluble MET kinase protein produced. Of 10 N-terminal boundariestested, residue Gly 1056 was chosen because it showed the most (albeitweak) soluble expression with the least (albeit large) amount ofinsoluble expression. In the second step residue Gly 1056 was keptconstant while four alternative C-termini were compared. With Gly 1056as the N-terminal boundary and Gly 1364 as the C-terminal boundary,soluble active MET kinase protein was generated at levels useful forbiochemical assays and crystallization.

To produce the various kinase domains described above, the completeintracellular domain of c-MET was cloned using a standard PCR reactionusing human brain cDNA (Invitrogen) as a template mixture and primersMet 950 and Ser-1408 (see table below). This complete domain was clonedinto a His-tagged vector, pET-N6 that is a derivative of pET24(Novagen). The pET-N6 vector is modified to encode a His tag (MGHHHHHHM)(SEQ ID NO: 4) that is fused to the N-terminus of coding sequencesengineered after it, and also includes a polylinker with NdeI, NotI,SalI, and EcoRI sites. Standard PCR reactions were used to engineer thevarious lengths of MET, using appropriate combinations of two primersfrom the table below.

TABLE 1 Starting amino acid residue SEQ ID NO DNA oligonucleotideprimer - Coding strand Met 950 5′-GAATTAGTT CATATGGATGCAAGAGTACACACTCCTCA  5 Lys 974 5′-TTTCCTG CATATGAAAAAGAGAAAGCAAATTAAAGATCT  6 Met 1013 5′-AACTACA CATATGGTTTCAAAATGAATCTGTAGACTAC  7 Gly 1037 5′-TTCATCT CATATGGGTTCATGCCGACAAGTGCAG  8 Met 1049 5′-TCTGACA CATATG TCCCCCATCCTAACTAGTGG 9 Gly 1056 5′-CATCCTA CATATG GGGGACTCTGATATATCCAGTC 10 Ala 10755′-GCAGCTA CATATG GCTCTAAATCCAGAGCTGGTCAT 11 Glu 1079 5′-TGCTCTA CATATGGAGCTGGTCCAGGCAGTGCA 12 Ala 1083 5′-GCAGCTA CATATGGGAAGAGGGCATTTTGGTTGTGT 13 Pro 1063 5′-GCAGCTA CATATGCCATTACTGCAAAATACTGTCCAC 14 DNA oligonucleotide primer - Noncodingstrand Ile 1363 5′-GACAA GTCGAC TA AATGAAAGTAGAGAAGATCGCTG 15 Gly 13645′-CTAGCAG GTCGAC TA CCCAATGAAAGTAGAGAAGATCGC 16 Glu 1365 5′-CTAGCAGGTCGAC TA CTCCCCAATGAAAGTAGAGAAGAT 17 Ser 1408 5′-AGGATCC GTCGAC TATGATGTCTCCCAGAAGGAG 18

These were cloned into the pET-N6 vector and the pET-N6 BI-PTPbicistronic vector for MET expression alone, or with PTP1B. Whenexpressed alone the MET was phosphorylated and when coexpressed withPTP1B the MET was unphosphorylated, as determined using methods asdescribed for the Abl kinase domain. Both the phosphorylated andunphosphorylated forms of MET kinase had kinase activity in the sameassay format as described for Abl kinase.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, variations can be made to the particular enzymes or enzymepairs utilized. Thus, such additional embodiments are within the scopeof the present invention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

TABLE 2 pET-SPEC BI-PTP Abl G227-V515-X (SEQ ID NO: 19) (Encoded aminoacid sequences are disclosed as SEQ ID NOS 23-24)TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGTCACCACCATCACCACCACGGTGTGTCC                            M  G  H  H  H  H  H  H  G  V  SCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGC P  N  Y  D  K  W  E  M  E  R  T  D  I  T  M  K  H  K  L  GGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCC G  G  Q  Y  G  E  V  Y  E  G  V  M  K  K  Y  S  L  T  V  AGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTC V  K  T  L  K  E  D  T  M  E  V  E  E  F  L  K  E  A  A  VATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCC M  K  E  I  K  H  P  N  L  V  Q  L  L  G  V  C  T  R  E  PCCGTTCTATATCATCACTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAG P  F  Y  I  I  T  E  F  M  T  Y  G  N  L  L  D  Y  L  R  ETGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCA C  N  R  Q  E  V  N  A  V  V  L  L  Y  M  A  T  Q  I  S  SGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGC A  M  E  Y  L  E  K  K  N  F  I  H  R  D  L  A  A  R  N  CCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGTTGATGACA L  V  G  E  N  H  L  V  K  V  A  D  F  G  L  S  R  L  M  TGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAG G  D  T  Y  T  A  H  A  G  A  K  F  P  I  K  W  T  A  P  EAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTT S  L  A  Y  N  K  F  S  I  K  S  D  V  W  A  F  G  V  L  LTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTAT W  E  I  A  T  Y  G  M  S  P  Y  P  G  I  D  L  S  Q  V  YGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTAT E  L  L  E  K  D  Y  R  M  E  R  P  E  G  C  P  E  K  V  YGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTTTGCTGAAATC E  L  M  R  A  C  W  Q  W  N  P  S  D  R  P  S  F  A  E  ICACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAG H  Q  A  F  E  T  M  F  Q  E  S  S  I  S  D  E  V  E  K  ECTGGGGAAACAAGGCGTCTGAGTCGACGAAGGAGATATATCCATGGAGATGGAAAAGGAG L  G  K  Q  G  V  -                       M  E  M  E  K  ETTCGAGCAGATCGACAAGTCCGGGAGCTGGGCGGCCATTTACCAGGATATCCGACATGAA F  E  Q  I  D  K  S  G  S  W  A  A  I  Y  Q  D  I  R  H  EGCCAGTGACTTCCCATGTAGAGTGGCCAAGCTTCCTAAGAACAAAAACCGAAATAGGTAC A  S  D  F  P  C  R  V  A  K  L  P  K  N  K  N  R  N  R  YAGAGACGTCAGTCCCTTTGACCATAGTCGGATTAAACTACATCAAGAAGATAATGACTAT R  D  V  S  P  F  D  H  S  R  I  K  L  H  Q  E  D  N  D  YATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGGAGTTACATTCTTACCCAGGGC I  N  A  S  L  I  K  M  E  E  A  Q  R  S  Y  I  L  T  Q  GCCTTTGCCTAACACATGCGGTCACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGGGT P  L  P  N  T  C  G  H  F  W  E  M  V  W  E  Q  K  S  R  GGTCGTCATGCTCAACAGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCA V  V  M  L  N  R  V  M  E  K  G  S  L  K  C  A  Q  Y  W  PCAAAAAGAAGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCT Q  K  E  E  K  E  M  I  F  E  D  T  N  L  K  L  T  L  I  SGAAGATATCAAGTCATATTATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAA E  D  I  K  S  Y  Y  T  V  R  Q  L  E  L  E  N  L  T  T  QGAAACTCGAGAGATCTTACATTTCCACTATACCACATGGCCTGACTTTGGAGTCCCTGAA E  T  R  E  I  L  H  F  H  Y  T  T  W  P  D  F  G  V  P  ETCACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGCCCG S  P  A  S  F  L  N  F  L  F  K  V  R  E  S  G  S  L  S  PGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTGGAACCTTCTGT E  H  G  P  V  V  V  H  C  S  A  G  I  G  R  S  G  T  F  CCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGATATC L  A  D  T  C  L  L  L  M  D  K  R  K  D  P  S  S  V  D  IAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGCCGACCAG K  K  V  L  L  E  M  R  K  F  R  M  G  L  I  Q  T  A  D  QCTGCGCTTCTCCTACGTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGTGAGAATTC L  R  F  S  Y  L  A  V  I  E  G  A  K  F  I  M  G  -GSGGCCAGCAGGGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGTCTTGAGGGGTTTTTTG

TABLE 3 Homo sapiens protein tyrosine phosphatase, non-receptor type 1(PTPN1), mRNA 3318 bp linear. ACCESSION NUMBER NM_002827 REFERENCE 1(bases 1 to 3318); Sun et al. (2003) J. Biol. Chem. 278: 12406-12414,Crystal structure of PTO1B complexed with a potent and selectivebidentate inhibitor REFERENCE 2 (bases 1 to 3318); Boute et al. (2003)EMBO Rep. 4: 313-319. REFERENCE 17 (bases 1 to 3318); Charbonneau et al.(1989) Human placenta protein-tyrosine-phosphatase: amino acid sequenceand relationship to a family of receptor-like proteins, Proc. Natl.Acad. Sci. USA 86: 5252-5256. The protein encoded by this gene is thefounding member of the protein tyrosine phosphatase (PTP) family, whichwas isolated and identified based on its enzymatic activity and aminoacid sequence. PTPs catalyze the hydrolysis of the phosphate monoestersspecifically on tyrosine residues. Members of the PTP family share ahighly conserved catalytic motif, which is essential for the catalyticactivity. PTPs are known to be signaling molecules that regulate avariety of cellular processes including cell growth, differentiation,mitotic cycle, and oncogenic transformation. This PTP has been shown toact as a negative regulator of insulin signaling by dephosphorylatingthe phosphotryosine residues of insulin receptor kinase. This PTP wasalso reported to dephosphorylate epidermal growth factor receptorkinase, as well as JAK2 and TYK2 kinases, which im- plicated the role ofthis PTP in cell growth control, and cell response to interferonstimulation. (SEQ ID NO: 20) translation= “MEMEKEFEQIDKSCSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMCDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEKGSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDEDHALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT” base pairs 181 . . . 1011encode Protein tyrosine phosphatase catalytic domain (SEQ ID NO: 21)   1 gtgatgcgta gttccggctg ccggttgaca tgaagaagca gcagcggcta gggcggcggt  61 agctgcaggg gtcggggatt gcagcgggcc tcggggctaa gagcgcgacg cggcctagag 121 cggcagacgg cgcagtgggc cgagaaggag gcgcagcagc cgccctggcc cgtcatggag 181 atggaaaagg agttcgagca gatcgacaag tccgggagct gggcggccat ttaccaggat 241 atccgacatg aagccagtga cttcccatgt agagtggcca agctccctaa gaacaaaaac 301 cgaaataggt acagagacgt cagtcccttt gaccatagtc ggattaaatt acatcaagaa 361 gataatgact atatcaacgc tagtttgata aaaatggaag aagcccaaag gagttacatt 421 cttacccagg gccctttgcc taacacatgc ggtcactttt gggagatggt gtgggagcag 481 aaaagcaggg gtgtcgtcat gctcaacaga gtgatggaga aaggttcgtt aaaatgcgca 541 caatactggc cacaaaaaga agaaaaagag atgatctttg aagacacaaa tttgaaatta 601 acattgatct ctgaagatat caagtcatat tatacagtgc gacagctaga attggaaaac 661 cttacaaccc aagaaactcg agagatctta catttccact ataccacatg gcctgacttt 721 ggagtccctg aatcaccagc ctcattcttg aactttcttt tcaaagtccg agagtcaggg 781 tcactcagcc cggagcacgg gcccgttgtg gtgcactgca gtgcaggcat cggcaggtct 841 ggaaccttct gtctggctga tacctgcctc ttgctgatgg acaagaggaa agacccttct 901 tccgttgata tcaagaaagt gctgttagaa atgaggaagt ttcggatggg gctgatccag 961 acagccgacc agctgcgctt ctcctacctg gctgtgatcg aaggtgccaa attcatcatg1021 ggggactctt ccgtgcagga tcagtggaag gagctttccc acgaggacct ggagccccca1081 cccgagcata tccccccacc tccccggcca cccaaacgaa tcctggagcc acacaatggg1141 aaatgcaggg agttcttccc aaatcaccag tgggtgaagg aagagaccca ggaggataaa1201 gactgcccca tcaaggaaga aaaaggaagc cccttaaatg ccgcacccta cggcatcgaa1261 agcatgagtc aagacactga agttagaagt cgggtcgtgg ggggaagtct tcgaggtgcc1321 caggctgcct ccccagccaa aggggagccg tcactgcccg agaaggacga ggaccatgca1381 ctgagttact ggaagccctt cctggtcaac atgtgcgtgg ctacggtcct cacggccggc1441 gcttacctct gctacaggtt cctgttcaac agcaacacat agcctgaccc tcctccactc1501 cacctccacc cactgtccgc ctctgcccgc agagcccacg cccgactagc aggcatgccg1561 cggtaggtaa gggccgccgg accgcgtaga gagccgggcc ccggacggac gttggttctg1621 cactaaaacc catcttcccc ggatgtgtgt ctcacccctc atccttttac tttttgcccc1681 ttccactttg agtaccaaat ccacaagcca ttttttgagg agagtgaaag agagtaccat1741 gctggcggcg cagagggaag gggcctacac ccgtcttggg gctcgcccca cccagggctc1801 cctcctggag catcccaggc gggcggcacg ccaacagccc cccccttgaa tctgcaggga1861 gcaactctcc actccatatt tatttaaaca attttttccc caaaggcatc catagtgcac1921 tagcattttc ttgaaccaat aatgtattaa aattttttga tgtcagcctt gcatcaaggg1981 ctttatcaaa aagtacaata ataaatcctc aggtagtact gggaatggaa ggctttgcca2041 tgggcctgct gcgtcagacc agtactggga aggaggacgg ttgtaagcag ttgttattta2101 gtgatattgt gggtaacgtg agaagataga acaatgctat aatatataat gaacacgtgg2161 gtatttaata agaaacatga tgtgagatta ctttgtcccg cttattctcc tccctgttat2221 ctgctagatc tagttctcaa tcactgctcc cccgtgtgta ttagaatgca tgtaaggtct2281 tcttgtgtcc tgatgaaaaa tatgtgcttg aaatgagaaa ctttgatctc tgcttactaa2341 tgtgccccat gtccaagtcc aacctgcctg tgcatgacct gatcattaca tggctgtggt2401 tcctaagcct gttgctgaag tcattgtcgc tcagcaatag ggtgcagttt tccaggaata2461 ggcatttgcc taattcctgg catgacactc tagtgacttc ctggtgaggc ccagcctgtc2521 ctggtacagc agggtcttgc tgtaactcag acattccaag ggtatgggaa gccatattca2581 cacctcacgc tctggacatg atttagggaa gcagggacac cccccgcccc ccacctttgg2641 gatcagcctc cgccattcca agtcaacact cttcttgagc agaccgtgat ttggaagaga2701 ggcacctgct ggaaaccaca cttcttgaaa cagcctgggt gacggtcctt taggcagcct2761 gccgccgtct ctgtcccggt tcaccttgcc gagagaggcg cgtctgcccc accctcaaac2821 cctgtggggc ctgatggtgc tcacgactct tcctgcaaag ggaactgaag acctccacat2881 taagtggctt tttaacatga aaaacacggc agctgtagct cccgagctac tctcttgcca2941 gcattttcac attttgcctt tctcgtggta gaagccagta cagagaaatt ctgtggtggg3001 aacattcgag gtgtcaccct gcagagctat ggtgaggtgt ggataaggct taggtgccag3061 gctgtaagca ttctgagctg ggcttgttgt ttttaagtcc tgtatatgta tgtagtagtt3121 tgggtgtgta tatatagtag catttcaaaa tggacgtact ggtttaacct cctatccttg3181 gagagcagct ggctctccac cttgttacac attatgttag agaggtagcg agctgctctg3241 ctatatgcct taagccaata tttactcatc aggtcattat tttttacaat ggccatggaa3301 taaaccattt ttacaaaa

TABLE 4 Serine/threonine protein phosphatase from Bacteriophage lambda.ACCESSION P03772; 221 aa (residues 1-221) Genomic sequence ofbacteriophage lambda is available at Accession numbes J02459 M17233M24325 V00636 X00906. REFERENCE 1 (residues 1 to 221); Sanger eta>(1982) Nucleotide sequence of bacteriophage lambda DNA, J. Mol. Biol.162 (4), 729-773. amino acid residues 1-221 of lambda serine/threonineprotein phosphatase (SEQ ID NO: 22)   1 mryyekidgs kyrniwvvgd lhgcytnlmnkldtigfdnk kdllisvgdl vdrgaenvec  61 lelitfpwfr avrgnheqmm idglsergnvnhwllngggw ffnldydkei lakalahkad 121 elpliielvs kdkkyvicha dypfdeyefgkpvdhqqviw nrerisnsqn givkeikgad 181 tfifghtpav kplkfanqmy idtgavfcgnltliqvqgeg a

1. A method for expressing a recombinant tyrosine kinase domain withreduced phosphorylation, said method comprising co-expressing saidrecombinant tyrosine kinase domain with a recombinant protein tyrosinephosphatase-1b (PTP1b) phosphatase domain that removes phosphate groupsfrom residues of said tyrosine kinase domain.
 2. The method of claim 1,wherein said tyrosine kinase domain and said PTP1b phosphatase domainare expressed in a cellular expression system.
 3. The method of claim 2,wherein said tyrosine kinase domain and said PTP1b phosphatase domainare expressed from a bi-cistronic mRNA.
 4. The method of claim 2,wherein said tyrosine kinase domain and said PTP1b phosphatase domainare expressed linked as a single fusion protein.
 5. The method of claim2, wherein said tyrosine kinase domain and said PTP1b phosphatase domainare encoded by a single vector.
 6. The method of claim 2, wherein saidtyrosine kinase domain and said PTP1b phosphatase domain are encoded byseparate vectors.
 7. The method of claim 1, wherein said tyrosine kinaseautophosphorylates.
 8. The method of claim 2, wherein said tyrosinekinase comprises the human c-MET kinase domain.
 9. The method of claim2, wherein said tryosine kinase comprises the human c-Abl kinase domain.10. A method for expressing a tyrosine kinase enzyme having reducedphosphorylation comprising co-expressing a recombinant tyrosine kinaseenzyme with a recombinant protein tyrosine phosphatase-1b (PTP1b) enzymethat dephosphorylates said tyrosine kinase in a cell.
 11. The method ofclaim 10, wherein said tyrosine kinase and said PTP1b are expressed froma bi-cistronic mRNA.
 12. The method of claim 10, wherein said tyrosinekinase and said PTP1b are expressed linked as a single fusion protein.13. The method of claim 10, wherein said tyrosine kinase and said PTP1bare encoded by a single vector.
 14. The method of claim 10, whereintyrosine kinase and said PTP1b are encoded by separate vectors.
 15. Themethod of claim 10, wherein said tyrosine kinase autophosphorylates. 16.The method of claim 10, wherein said recombinant tyrosine kinase enzymeis modified by an endogenous enzyme produced by the cell used forexpression of said tyrosine kinase enzyme.
 17. A cell comprising: afirst recombinant nucleic acid sequence encoding a tyrosine kinaseenzyme subject to enzymatic phosphorylation; and a second recombinantnucleic acid sequence encoding a protein tyrosine phosphatase-1b (PTP1b)enzyme that reverses said phosphorylation, wherein said first and secondrecombinant nucleic acid sequences are operatively linked withregulatory sequence(s) such that said first and second recombinantnucleic acids are expressed in said cell.
 18. The cell of claim 17,wherein said tyrosine kinase enzyme autophosphorylates when expressed inthe cell.
 19. The cell of claim 17, wherein said cell is from an E. colistrain.
 20. The cell of claim 17, wherein said tyrosine kinase enzymeconsists essentially of a tyrosine kinase catalytic domain and saidPTP1b enzyme consists essentially of a PTP1b catalytic domain, whereinsaid PTP1b catalytic domain reduces the level of phosphate groupmodification on said tyrosine kinase catalytic domain.
 21. The cell ofclaim 7, wherein said tyrosine kinase and said PTP1b are expressedlinked as a single fusion protein.
 22. The cell of claim 17, whereinsaid tyrosine kinase enzyme and PTP1b enzyme are expressed from abi-cistronic mRNA.
 23. The cell of claim 17, wherein said tyrosinekinase enzyme and PTP1b enzymes are expressed from a single vector. 24.The cell of claim 17, wherein said tyrosine kinase enzyme and PTP1benzymes are expressed from separate vectors.